Formation of bilayers of amphipathic molecules

ABSTRACT

A method of forming bilayers of amphipathic molecules uses droplets of aqueous solution in a hydrophobic medium such as oil. A layer of amphipathic molecules such as a lipid is formed around the surfaces of the droplets. This may be achieved by providing the lipid in the oil and leaving the droplets for a time sufficient to form the layer. The droplets are brought into contact with one another so that a bilayer of the amphipathic molecules is formed as an interface between the contacting droplets. The bilayers may be used for a wide range of studies. The technique has numerous advantages including providing a long lifetime for the bilayers, allowing study of small volumes and allowing the construction of chains and networks of droplets with bilayers in between to study complex systems.

This application is a continuation of U.S. application Ser. No.12/375,103, filed Sep. 9, 2009 now U.S. Pat. No. 8,268,627, which is theU.S. national phase of International Application No. PCT/GB2007/002856,filed Jul. 26, 2007, which designated the U.S. and claims priority toGreat Britain Application No. 0614835.7, filed Jul. 26, 2006, the entirecontents of each of which are hereby incorporated by reference.

This invention was made with government support under Grant No.FA9550-06-C-0006 awarded by USAF/AFOSR. The government has certainrights in this invention.

The present invention relates to the formation of bilayers ofamphipathic molecules such as lipids. Such bilayers are models of cellmembranes and as such may be used to perform a wide range of studies inbiotechnology.

Lipid bilayers, or more generally bilayers of amphipathic molecules, aremodels of cell membranes and serve as excellent platforms for a range ofexperimental studies, for example in vitro investigation of membraneproteins by single-channel recording. Conditions such as temperature,bilayer composition (surface charge, cholesterol content etc.) andtransmembrane potential can be adjusted to either mimic biologicalsystems or venture beyond the physiological range. Most importantly,manipulation of the solution conditions (pH, salt composition, ionicstrength) is possible on both sides of the membrane. While the in vivoinvestigation of membrane proteins in living cells is possible by thepatch clamp method, the conditions that can be used are limited by therequirement to keep the cells healthy.

In the conventional technique, planar lipid bilayers are formed over aplastic aperture which has been pretreated with an oily mixture. Despitewidespread use in academic research, this conventional technique suffersfrom a number of limitations, for example as follows.

The conventional technique is cumbersome and the bilayers produced aredelicate. The two most common methods of forming planar bilayers are topaint a lipid/oil mixture across an aperture and to fold together twomonolayers, one from each side of an aperture. Both techniques requirethe hands of a skilled scientist. Small hydrostatic forces due tovibration or flow often rupture planar bilayers. Even under the bestconditions, the lifetime of a planar bilayer is usually only a fewhours. The limited lifetime effectively limits the range of processeswhich it is possible to study.

Also, since the size and shape of the each bilayer's annulus region(which governs the membrane's properties) is unique, the insertion ofmembrane proteins into planar bilayers is difficult to standardize.

Another cause of difficulty when studying some systems is that thevolume of the electrical recording cell on either side of the planarbilayer is typically greater than 1 mL. Therefore, each experiment needslarge amounts of protein or other reagents, which may not be easy toproduce. After each experiment, both sides of the electrical recordingcell must be thoroughly cleaned.

Lastly, the conventional technique uses a single planar bilayer betweentwo chambers. To study systems comprising larger numbers of membranes,it would in principle be possible to construct a cell having morechambers and membranes, but the experimental techniques of forming thebilayers would be very complicated and in practical terms would makethis very labourious and unattractive to the scientist.

It would be desirable to develop an alternative method which alleviatesone or more of these limitations.

According to the present invention, there is provided a method offorming bilayers of amphipathic molecules, comprising:

forming a plurality of droplets of aqueous solution in a hydrophobicmedium with a layer of amphipathic molecules around the surfaces of thedroplets;

bringing droplets into contact with one another so that a bilayer of theamphipathic molecules is formed as an interface between contactingdroplets.

This method of forming bilayers has considerable advantages and inparticular overcomes the limitations of the conventional techniquediscussed above.

The method is straightforward to perform and results in robust bilayerswhich can be used in a wide range of studies and applications in thefield of biotechnology. The droplets can be formed very easily, forexample simply by pipetting aqueous solution into the hydrophobicmedium. Similarly, the droplets are easily moved around to bring theminto contact with each other. Indeed the manner in which droplets aremoved is not critical as the droplets are robust and easily manipulated.Examples of suitable apparatus for handling the droplets are givenbelow, but are not limitative.

The formation of a layer of amphipathic molecules around the surfaces ofthe droplets is also straightforward. For example, it may be achievedsimply by providing the amphipathic molecules in the hydrophobic mediumor in the aqueous solution of the droplets, whereupon the layer can formnaturally if the droplets are left for a sufficient period of time. Theamphipathic molecules may also be dissolved, or suspended as lipidvesicles in the droplets themselves, from where they again spontaneouslyform monomolecular layers at the interface between the droplet and thehydrophobic medium, that may have an equilibrating concentration of theamphipathic molecule in the hydrophobic medium.

The bilayer is formed simply by bringing droplets into contact with oneanother. The orientation of the amphipathic molecules in the layeraround the aqueous solution allows the formation of the bilayer. As thedroplets are brought into contact, after the intervening hydrophobicmedium has been displaced the bilayer forms very quickly as an interfacebetween the contacting droplets. The bilayer forms a planar surfacebetween the two droplets which are otherwise generally spherical. Thisplanar bilayer is the shape with the lowest free surface energy and hasa negative free energy of formation; it is therefore a spontaneousevent. The amphipathic molecules allow two droplets to be brought intocontact without allowing them to coalesce by the formation of a stablebilayer.

The droplets may be handled by a variety of techniques. One particularlyadvantageous method of moving the droplets is to dispose an anchorhaving a hydrophilic outer surface inside a droplet. Movement of theallows the droplet to be moved, for example to bring it into contactwith another droplet.

The bilayer can be used to perform experiments involving a processoccurring at or through the bilayer of the amphipathic molecules. Amajor class of experiments use a membrane protein inserted into thebilayer. This may be achieved simply by providing the membrane proteinin the aqueous solution. It has been shown that after the formation ofthe bilayer, the membrane protein naturally inserts into the bilayer inthe same manner as with a bilayer formed by the conventional technique.

It has been observed that the bilayer behaves functionally in the samemanner as a bilayer formed by the conventional technique. Therefore thebilayer formed by the present method can be used to perform the sametypes of experiments, but providing a number of advantages which broadenthe range of possible experiments, as discussed further below. Thus thepresent method may be applied to a wide range of experiments includinginvestigation and/or screening of membrane proteins, investigationand/or screening of analytes which interact with membrane proteins, andinvestigation and/or screening of the bilayers. Indeed the method may beused to study any bilayer phenomena in general, typically involving aprocess occurring at or through the bilayer.

The lipid bilayer may also be used to study the properties of themembrane protein inserted therein. For example, the voltage dependenceof the properties of the membrane protein may be determined Techniquesfor studying membrane proteins in lipid bilayers are well known in theart. The function of a channel or pore may be determined by measuring,for example, an ionic current flowing across the lipid bilayer through amembrane protein. The function of a transporter may be determined bymeasuring the amount of a molecule translocated across the lipidbilayer, for example by mass spectrometer or ELISA or by using asubstrate which is tagged fluorescently or radioactively.

Other examples of experiments which may be performed as follows.

The water droplets can be osmotically inflated or deflated depending ontheir initial osmolarity. Water can transfer between the droplets inresponse to an osmotic gradient through the formed bilayer at thecontact. Also, other molecules that are bilayer permeable, like drugs,or imageable molecules can be made to move from one droplet to anotherthrough the bilayer contact zone. Thus, reactants can be separated andallowed to react only when transported across the bilayer. Hereapplications may involve microfluidic systems of droplets that can bebrought into contact and allowed to react to produce new products formedonly when the reactants cross the bilayer contacts between differentdroplets. One possibility is that one reactant is bilayer permeablewhile the other is not bilayer permeable. In this case the products onlyoccur in the droplet containing the non-bilayer permeable reactant. Ifon the other hand both reactants are bilayer transferable then theproducts can be formed in both droplets, or a plurality of contactingdroplets, dependent on the relative permeability of each reactant acrossthe contact bilayers. These examples do not involve membrane proteinsper se, but just the formed bilayer contacts through which reactants andproducts might diffuse.

Further specific examples of studies to which the present method may beapplied are discussed further below.

In many studies electrical measurements are taken. This isstraightforward to achieve by bringing electrodes into electricalcontact with the droplets when the droplets are in contact with oneanother, for example by insertion of the electrodes into the droplets orby placing droplets onto static electrodes inserted into the chamber orin the microfluidic channels

Bilayers formed by the present method have the advantage of being robustand having a long lifetime, as compared to the conventional technique.For example bilayers formed by the conventional technique require skillto prepare and typically last a few hours and at most in a very smallpercentage of cases a couple of days. In contrast bilayers formed by thepresent technique are formed more reliably and last much longer,generally lasting a number of days. Although a full study of lifetimehas not been performed, a bilayer has been observed to last for a periodof 8 days before it was purposely divided by separating the drops.

It is hypothesised that the reason for the higher lifetime is that thebilayer formed between two droplets has a lower surface free energy thana bilayer formed by the conventional technique. In the latter case, inthe annulus region adjacent the periphery of the aperture, the bilayerdivides into two monolayers which extend on opposite sides of thebarrier defining the aperture. It may also be that stability isconferred by the absence of an annulus that has to be attached (oradsorbed) onto the material of a septum defining an aperture. In asimilar fashion to the conventional technique, the bilayer also dividesinto two monolayers which in this case simply coat the dropletinterfaces, and do not terminate at a support material (except at thesurface of the chamber). The bilayer is therefore not “stretched” acrossan aperture, but forms at the contact zone of the two droplets and noadditional spreading or wetting tensions are induced, just the bilayertension determined by the monolayer tension and the free energy offormation in the hydrophobic medium.

The higher lifetime allows the study of biological processes whichthemselves have a longer lifetime. In this way the present method opensup new fields of study.

The formation of the bilayers is also highly reversible and repeatable.Droplets which have been brought into contact with one another may befreely separated to divide the bilayer and may be subsequently broughtinto contact again to re-create the bilayer. Such control over thecreation, division and re-creation of bilayers also opens up new fieldsof study.

The degree of control makes the formation of the bilayers easy tostandardise. In particular, it is easy to vary the area of the bilayerof the amphipathic molecules by moving the droplets when the dropletsare in contact with one another. The change in the area of the bilayersmay be observed visually or by capacitance measurements. It has beendemonstrated that it is possible to change the diameter of the bilayerover the range from 30 μm to 1000 μm, although this is not thought to bethe limit.

In addition, the nature of the hydrophobic medium determines the degreeof spreading of the contacted monolayers and thereby the contact angle.For example, in experiments it has been observed that for bilayers ofglycerylmonooleate (GMO) formed in decane as the hydrophobic medium, thecontact area is relatively small and the contact angle is about 3°, thisbeing in agreement with contact angles measured in conventional lipidmembrane systems. On the other hand, if the hydrophobic medium issqualene, a larger contact area is formed and the contact angle is 25°,again in agreement with measurements on conventional lipid membranes.These solvent-dependent effects reflect the small free energy offormation of the GMO:decane system (around −4 mJ/m²) as compared to theGMO:squalene system (around −500 mJ/m²), where the bilayer thicknessconcomitantly decreased from 50 Å to 25 Å, signifying a depletion of thelarger squalene solvent from the bilayer. This non-linear increase infree energy of formation departs from simple Lifshitz theory for twoinfinite slabs of water acting across the thin oil film, and is more inline with a “depletion flocculation” effect. Essentially, the largersqualene solvent molecules are entropically excluded from the GMObilayer, and this depletion of solvent exerts a greater osmotic pressureon the bilayer, thereby raising the free energy of formation by ordersof magnitude in going from decane to squalene, over and above anyLifshitz effects. Adhesion and the strength and stability of the contactthen are largely dependent on the presence or absence of solvent in thebilayer.

Another advantage of the present method is that it allows the use of arelatively small volume of aqueous solution. In particular, the volumemay be smaller than that present in the chambers of a cell used in theconventional technique. The droplets may typically have a volume lessthan 1000 nL. In general the droplets may be of any size limited only bythe degree of control of the dispenser of the aqueous solution and thelimits of optical resolution if direct manipulation is desired. Dropletsthat are not required to have electrical recording or stimulus fromplaced electrodes can be assembled in suspension forming a raft or 3Daggregate or flocculent of droplets having dimensions of micrometers toeven nanometers that are all in contact with each other via theirintervening bilayers. Using a standard pipette, experiments have beenperformed on droplets having volumes in the range from 200 nL to 800 nLbut it is expected that droplets of smaller volumes could be producedwith suitable equipment. For example, using micro-pipette manipulationto form the droplets from glass micro-pipettes observed in a relativelypowerful microscope, where droplets of diameter say 30 μm are assembled,the volume is approximately 14 pL. In suspension, droplet aggregation ofdroplets of diameter say 200 nm yields internal volumes of approximately4 aL (aL stands for attoliter being 10⁻¹⁸ L).

An important advantage of the present invention is that it is possibleto bring more than two droplets into contact with each other in a chainor network, for example on a flat or dimpled surface, in a microfluidicchannel or, as alluded to above, in aggregated or flocculatedsuspension. The simplicity and control with which the bilayers can beformed simply by moving droplets around makes it straightforward tobuild large chains or networks which would be impractical in a systemwhere bilayers are formed in apertures in barriers in accordance withthe conventional technique. This opens up the possibility of studyingmuch larger systems than is practical with the conventional technique,for example modelling entire systems using multiple droplets. Someexamples are given below, but the range of science which could bestudied is much wider.

The method can be performed with a wide range of materials, as follows.

In general, the amphipathic molecules can be of any type which form abilayer in the hydrophobic medium in which the droplets are positioned.This is dependent on the nature of the hydrophobic medium and theaqueous solution, but a wide range of amphipathic molecules arepossible. Amphipathic molecules are molecules which have bothhydrophobic and hydrophilic groups. The layer formed around the dropletis a monolayer of amphipathic molecules which is formed and maintainednaturally by the interaction of the hydrophobic and hydrophilic groupswith the aqueous solution so that the molecules align on the surface ofthe droplet with the hydrophilic groups facing inwards and thehydrophobic groups facing outwards.

An important class of amphipathic molecules to which the present methodmay be applied is lipid molecules. The lipid molecules may be any of themajor classes of lipid, including fatty acyls, glycerolipids,glycerophospholipids, sphingolipids, sterol lipids, prenol lipids,saccharolipids and polyketides. Some important examples include aphospholipid, a glycolipid or cholesterol. The lipid molecules may benaturally occurring or synthetic. Whilst the formation of a bilayer fromlipid molecules has been demonstrated the method is expected to beappropriate for any amphipathic molecules capable of forming a bilayer.

The amphipathic molecules need not be all of the same type. Theamphipathic molecules may be mixtures. Another important example is thatthe amphipathic molecules in the respective layers of two dropletsbrought into contact are of different types so that the bilayer formedby the two monolayers is asymmetric.

The aqueous solution may be freely chosen for the experimental studywhich is to be performed. The aqueous solution of each droplet may bethe same or different. The nature and concentration of the solutes canbe freely varied to vary the properties of the solution. One importantproperty is pH and this can be varied over a wide range. Anotherimportant point in experiments using electrical measurements is toselect appropriate salts to carry the current. Another importantproperty is osmolarity.

The hydrophobic medium can also be selected from a wide range ofmaterials. The material is hydrophobic so that the aqueous solutionforms a droplet rather than mixing with the hydrophobic medium butotherwise the hydrophobic medium can be freely chosen. The viscosity ofthe hydrophobic medium can be selected to affect the movement of thedroplets and the speed of formation of the layer of amphipathicmolecules in the case that they are provided in the hydrophobic medium.

The hydrophobic medium may be an oil. Any type of oil is suitable aslong as its surface activity is relatively high, and that it does notdestabilize the formed bilayers. The oil may be a hydrocarbon which maybe branched or unbranched, for example a hydrocarbon having from 5 to 20carbon atoms (although hydrocarbons of lower molecule weight wouldrequire control of evaporation). Suitable examples include alkanes oralkenes, such as hexadecane, decane, pentane or squalene. Other types ofoil are possible. For example the oil may be a fluorocarbon. This mightbe useful for the study of some systems, for example to minimise loss ofa particular membrane protein or analyte from the droplet or to controlgas content such as oxygen.

As discussed above, in many experimental studies a membrane protein isprovided in one or more of the droplets for insertion into the bilayer.The present method does not limit the choice of membrane protein,provided that the aqueous solution is chosen with appropriate propertiesfor the protein in question. Thus the membrane protein may be of anytype. The use of integral membrane proteins has been demonstrated, butit is equally expected that peripheral membrane proteins could be used.The present method applies to any membrane proteins including the twomajor classes that is β-barrels or α-helical bundles. An importantapplication is a membrane protein which is a pore or a channel. Besidesa protein pore or channel, further possible membrane proteins include,but not exclusively, a receptor, a transporter or a protein whicheffects cell recognition or a cell-to-cell interaction.

To allow better understanding, embodiments of the present invention willnow be described by way of non-limitative example with reference to theaccompanying drawings, in which:

FIG. 1 is a cross-sectional view of an apparatus for handling dropletsto form a bilayer;

FIG. 2 is a schematic cross-sectional view of two contacting dropletswith a bilayer formed as an interface therebetween;

FIG. 3 is a cross-sectional view of an alternative apparatus forhandling droplets to form a bilayer;

FIGS. 4 to 7 are traces of the ionic current in particular experimentsperformed using bilayers formed between two droplets.

FIG. 8 is an image of a network of droplets;

FIG. 9 is a diagram of a network of droplets forming a “bio-battery”;

FIG. 10 is a trace of current recorded in the network of FIG. 9;

FIG. 11 is a diagram of a network of droplets having light sensingcapabilities; and

FIG. 12 is a trace of current recorded in the network of FIG. 11.

An apparatus 1 which is suitable for handling droplets of aqueoussolution to perform the present method is shown in FIG. 1. The apparatus1 comprises a container 2 being a 1 mL Perspex bath. The container 2contains an oil 3.

Within the oil 3 are two droplets 4 of aqueous solution. As the oil 3 isa hydrophobic medium, the aqueous solution of the droplets 4 does notmix with the oil 3 to any large extent. Some mutual solubility isexpected depending on the solubility limit of a hydrocarbon in the waterand the water in the hydrocarbon. The droplets 4 are formed into the oil3 simply by dispensing the aqueous solution, for example from aconventional pipette or indeed any suitable dispenser. The dispenser ispreferably of a type which allows the volume of the droplets 4 to becontrolled. Experiments have been performed using droplets 4 of volumein the range from 200 nL to 800 nL but this is not limitative. One ofthe advantages of the method is the ability to use droplets 4 of smallvolume and it is expected that volumes less than 200 nL could be used.Micro-pipette manipulation has been used to make droplets 4 of only tensof micrometers and it is anticipated that emulsification techniques canproduce suspensions of bilayer contacting droplets that have diametersof order 100 nm.

On the base of the container 2 within the oil 3, the apparatus 1 isprovided with three supports 5 having a hydrophobic outer surface. Thesupports 5 are in this example simply 10 μL disposable pipette tipsmounted on the container 1. Thus the supports 5 are annular. Thedroplets 4 may be disposed on the supports 5 by dispensing them in thislocation. By way of example, FIG. 1 shows a droplet 4-A supported on oneof the supports 5. The hydrophobic nature of the outer surface of thesupport 5 which arises due to the support 5 being made of plasticprevents the droplet 4-A supported thereon from flowing down over thesupport 5. As an alternative there could be used a support in the formof a dimpled surface.

Each support 5 is provided with an anchor 6 formed by a hydrogel droplet7 held on the end of an electrode 8 formed by a 100 μm diameter rodcoated with Ag/AgCl which protrudes 0.5 mm through the aperture in thecentre of the support 5 so that the hydrogel droplet 7 is disposedinside the droplet 4-A of aqueous solution. In particular the hydrogelis 5% (w/v) agarose in buffer solution. Due to hydrogel having a veryhigh water content, the outer surface of the hydrogel droplet 7 ishydrophilic. As a result the hydrogel droplet 7 anchors the droplet 4-Asupported on the support 5 because of the attraction to the aqueoussolution of the droplet 4-A. In this way, the anchor 6 assists inholding the droplet 4-A on the support 5.

Further droplets 4, for example the droplet 4-B shown in FIG. 1, may bemoved around within the oil 3 using a micromanipulator 9 which is shownschematically in FIG. 1 and may be of a conventional type. The droplet4-B is held by an anchor 10 connected to the micromanipulator 9. Theanchor 10 comprises a section of Ag wire which has been partially meltedat the end to form a 200 μm diameter ball. This was first treated withNaClO to create an Ag/AgCl electrode 12 and then coated with a layer ofhydrogel to form a hydrogel droplet 11. In particular the hydrogel is 5%(w/v) agarose in buffer solution and has a thickness of order 200 μm.Due to hydrogel having a very high water content, the outer surface ofthe hydrogel droplet 11 is hydrophilic. As a result the hydrogel droplet11 anchors the droplet 4-B on the anchor 10 because of the attraction tothe aqueous solution of the droplet 4-B. Thus the droplet 4-B may bemoved around by controlling the micromanipulator 9 to move the anchor10.

After the droplets 4 of aqueous solution are formed in the oil 3, alayer of amphipathic molecules, such as lipid molecules, are formedaround the surfaces of the droplets 4. There are two options forachieving this.

The first option is to provide the amphipathic molecules in the oil 3.This may be done before dispensing the droplets 4 into the oil 3, thatis by providing the oil 3 as a solution of the amphipathic molecules inthe oil 3. Alternatively, the amphipathic molecules could be providedafter dispensing the droplets 4 into the oil 3 but in that case it isharder to mix the amphipathic molecules with the oil 3. The droplets 4themselves may contain the surfactant or the lipid in solution, micellarsuspension or as lipid vesicles or liposomes.

Subsequently, after the amphipathic molecules have been provided in theoil 3 and after dispensing the droplets 4 into the oil 3, a layer ofamphipathic molecules forms around the outer surface of the droplets 4spontaneously. This can be achieved simply by leaving the droplets 4 inthe oil 3 for a sufficient period of time. It is not necessary to takeany special measures to encourage formation of the layer, but measuressuch as agitation might speed up the process.

The second option is to provide the amphipathic molecules in the aqueoussolution dispensed into the oil 3 to form the droplets 4. For example,the amphipathic molecules may be provided as vesicles suspended in theaqueous solution. Subsequently, the layer of amphipathic molecules formsaround the outer surface of the droplets 4 spontaneously. This can beachieved simply by leaving the droplets 4 in the oil 3 for a sufficientperiod of time. It is not necessary to take any special measures toencourage formation of the layer, but measures such as agitation mightspeed up the process.

The period of time required to form the layer of amphipathic moleculesdepends on the nature of the oil 3, the amphipathic molecules and theaqueous solution, but is typically of the order of tens of minutes. Therequired time is easily determined experimentally for any given materialsystem. That is to say, trials can be performed in which droplets 4 areformed and left for different periods of time before the droplets 4 arebrought together to form a bilayer as described below. Trials where theperiod of time is too short will not result in a stable bilayer soinstead the droplets 4 merge together to form a larger droplet. Theperiod of time in respect of the trials where a stable bilayer does formis the appropriate period for the material system in question.

After the layer of amphipathic molecules has formed around the droplets4, the droplets 4 are brought into contact with one another. This isachieved in the apparatus of FIG. 1 by moving the droplet 4-B using themicromanipulator 9 until it is in contact with the static droplet 4-Asupported on the support 5.

When the droplets 4 are brought into contact a bilayer of theamphipathic molecules forms as the interface between the droplets 4.This is illustrated in FIG. 2 which shows two droplets 4 of aqueoussolution in the oil 3. Each droplet 4 is surrounded by a layer ofamphipathic molecules 13 which are shown schematically (and not toscale) oriented with their hydrophobic tails 13 a facing outwards andtheir hydrophilic heads 13 b facing inwards. Where the droplets 4 comeinto contact, the layers of amphipathic molecules 13 of each droplet 4form a bilayer 14. As it is the shape of lowest free surface energy, thebilayer 14 is planar, at least as compared to the monolayer ofamphipathic molecules 13 around the remainder of the droplets 4 (thebilayer 14 may have some small degree of curvature).

The formation of the bilayer occurs spontaneously when the droplets 4come into contact and may be observed visually through a microscope. Asthe droplets 4 come into contact there is a short delay where thedroplet 4 deform and then spontaneously the bilayer 14 forms with aplanar shape as shown in FIG. 2. The delay is the time taken for the oil3 between the droplets 4 to be displaced out of the interface.

The formation of the bilayer 14 can also be observed by electricalmeasurements, in particular of the capacitance between the droplets 4.To measure this and perform other electrical measurements, the apparatus1 further includes a circuit 15 of the same type as used in knownapparatuses for studying bilayers using the conventional methoddescribed above using an aperture in a barrier. The circuit 15 isconnected to the electrodes 8 and 12. An electrical contact with theaqueous solution of the droplets 4 is made due to the conductive natureof the hydrogel droplets 7 and 11. When a bilayer 14 forms, thecapacitance measured by the circuit 15 increases in the same manner aswith a bilayer formed by the conventional method.

A particular advantage of the present method is that the area of thebilayer 14 can be varied by moving the contacting droplets away andtowards each other. In the apparatus 1 of FIG. 1, this is achieved bymovement of the droplet 4-B by the micromanipulator 9. The changing areaof the bilayer 14 can be observed both visually and from the capacitancemeasurements. In experiments the average diameter of the bilayer 14 hasbeen changed in the range from 30 μm to 1000 μm although this is notlimitative. Capacitance measurement also allows for precise control ofthe area of the bilayer 14 which can provide the advantage that a givenexperiment can be standardised by using a bilayer 14 of standard area.Another advantage of varying the area is that insertion of a membraneprotein can be encouraged by initially forming a large bilayer 14 andafter insertion the area of the bilayer 14 can be reduced. It has beenobserved that during reduction of the area of the bilayer 14, themembrane protein remains inserted, as observed by measurements, untiljust before the bilayer 14 separates. Such reduction in the area of thebilayer 14 can also reduce noise in electrical measurements.

It has also been observed that the bilayer 14 can be repeatedly andreliably separated by separating the droplets 4 and re-created bybringing the droplets 4 back into contact. This is advantageous as itallows complex experiments to be performed.

The apparatus 1 is convenient but in general droplets 4 of aqueoussolution can be brought together to form a bilayer 14 in a wide range ofapparatuses. The droplets 4 themselves are sufficiently robust to bemanipulated in a number of different ways, for example by beingphysically pushed or placed, instead of anchoring them to an anchor.Similarly, the formation of the bilayer 14 is robust and repeatable notdependent on how the droplets 4 are manipulated. Thus a variety ofdifferent apparatuses can be used depending on the application. Themanipulation technique may be very simple. For example, the droplets 4may be moved simply by pushing them using a simple probe such as a glassor plastic rod. Alternatively, more complex manipulation techniques maybe applied. For example the droplets may be moved using micro-fluidicapparatus. There is extensive discussion in the literature ofmicro-fluidics being used to move droplets of aqueous solution in oiland such techniques may be advantageously applied to the presentinvention, for example to facilitate high through-put screening.

A simple, alternative apparatus 16 which has been used is shown in FIG.3. In this case, the base of the container 2, which in this case is madeof teflon, has electrodes 17 protruding through the base. The droplets 4are supported on the base enveloping the electrodes 17. The droplets 4are positioned simply by dispensing them in the oil 3 and subsequentlymanipulated by pushing them around. For example, droplets 4 may bedispensed onto the electrodes 17 or may be dispensed to a different areaof the base and pushed onto the electrodes 17. Although the apparatus 1of FIG. 1 is more convenient to use, the apparatus 16 of FIG. 3 doesallow bilayers 14 to be formed and monitored, thus demonstrating therobustness of the technique.

Conversely it is anticipated that other more complicated apparatusescould be used, depending on the application. For example for use inscreening, an array of droplets 4 might be supported and one or an arrayof further droplets 4 might be moved relative thereto. Another possibleway to move the droplets 4 would be to use micro-fluidics equipment orelectrical patterned equipment.

As already discussed, the bilayers 14 formed by the present method maybe used to perform a wide range of experiments involving a processoccurring at or through the bilayer of the amphipathic molecules. Someexamples of actual experiments which have been performed to demonstratethe efficacy of the present method are given below.

For example, having established that droplets 4 adhere and form abilayer 14 at the contact zone, it is of interest to measure the waterpermeability of the bilayer 14 such as in the GMO/solvent bilayersystem. This easily accomplished by simply forming two droplets 4 eachdroplet 4 having different osmotic pressure. In the experiment carriedout, we chose to assemble one droplet 4 of solution of 500 mOsm glucoseand the other droplet 4 of pure deionized water. The two micro-pipettesthat form the droplets 4 are filled each with the different solution,and using the pressure control, positive pressure is applied to firstone and then the other to generate the two droplets 4. Forming thedroplets 4 in GMO-squalene ensures that water is not rapidly lost to thesurrounding hydrocarbon phase and that changes in volume of the droplets4 represents the passage of water from the water droplet 4 to theglucose solution droplet 4 down the osmotic gradient, through thebilayer 14 formed at the contact. The experiment is then to simplyassemble the droplets 4 in to adherent contact and record on video theirprogress and volume changes (calculated from the diameters) that occurdue to water transport. The glucose solution droplet 4 takes up thewater from the pure water droplet 4 and grows as the water droplet 4shrinks Data is then plotted as droplet volume with time.

In many types of experiment, the physical process is monitored by takingelectrical measurements, eg of capacitance, current or voltage, usingthe circuit 15 in a similar manner to known techniques. To this end, thecircuit 15 may, by way of example, comprise a patch-clamp amplifier(Axopatch 200B; Axon Instruments), filtered with a low-pass Besselfilter (80 dB/decade) with a corner frequency of 2 kHz and thendigitized with a DigiData 1320 A/D converter (Axon Instruments) at asampling frequency of 5 kHz. The container 1 and amplifying headstage ofthe circuit 15 may be enclosed in a metal box to serve as a Faradaycage.

By way of example, the electrical measurement may measure the currentrepresentative of the passage of ions through a membrane protein whichis an ion channel. However, it is not essential to use electricalmeasurements, as any measurement characterising the process in questionmay be used. One alternative to electrical measurement is opticalmeasurement, for example of a fluorescent molecule which is transportedacross the bilayer 14 or which responds to another molecule transportedacross the bilayer 14 or to the potential across the bilayer 14.

Many experimental techniques involve the insertion of a membrane proteininto the bilayer 14, the process under study relating to the function ofthe membrane protein. In this case, the membrane protein may be used byproviding it in the aqueous solution of one or more of the droplets 4.The membrane protein then inserts spontaneously into the bilayer 14.This can be achieved simply by leaving the bilayer 14 until insertionoccurs without taking any special measures.

Above, only a single bilayer 14 between two droplets 4 has beenconsidered. However, it is a particular advantage of the present methodthat more than two droplets 4 may be brought into contact to form pluralbilayers 14 between the contacting droplets 4. The droplets 4 may bearranged in a chain or network. For example, in the apparatus 1 of FIG.1, three droplets 4 may be arranged on the three supports 5 with thedroplets 4 contacting each other in a chain, or alternatively droplets 4on the three supports 5 may be interconnected by other droplets 4 toform a longer chain. Such chains of droplets may be branched byconnecting further droplets 4 to the chain. Branches of droplets 4 maybe connected to other droplets 4 to form a network. It would bestraightforward to expand the apparatus 1 of FIG. 1 or indeed anyapparatus to accommodate large numbers of droplets 4 and moreelectrodes. Similarly in microfluidic channels two or more droplets 4can be conjoined to make a chain, or positioned in a network pattern ofchannels.

As the present method of forming bilayers 14 between droplets 4 isreliable and highly repeatable, this opens up the possibility of formingand studying complex systems of droplets 4 with bilayers 14 in betweenwhich would in practical terms be prohibitively difficult to do with theconventional method of forming bilayers across an aperture in a barrier.Furthermore, multiple interactions could be studied in sequence or inparallel. The possibilities are wide-ranging and exciting, and includemodelling of tissues such as heart tissue or any monolayer of epithelia,such as might exist in the gut, or retina, or ear where gap junctionsprovide cell to cell communication and redistribution of ions. Also,endothelial monolayers could be modelled.

Another possibility is for the aqueous solution of the droplets 4 toinclude solutes which cause transport through the membrane proteins,being either primary or secondary transport. In this way, the system maybe self-powered. Alternatively, the system may be externally powered,for example electrically by the circuit 15 or optically with lightactivated systems.

To illustrate the efficacy of the present method there will now bedescribed some experiments which have been performed using the apparatus1 of FIG. 1.

The first experiment demonstrates that ion-conducting membrane proteinsinsert in the bilayer 14 allowing measurements of ionic current to beperformed when a potential is applied across the bilayer 14.

In particular, two 200 nL droplets 4 were arranged on the support 5 andon the anchor 10, in both which the electrodes 8 and 12 were made ofAg/AgCl. The oil 3 was hexadecane (Sigma) and the lipid1,2-diphytanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPhPC, Avanti)was dissolved therein to form a 10 mM solution.

One droplet 4 contained an aqueous solution of 10 pg/ml wild-type (WT)staphylococcal α-hemolysin (αHL) heptamer in 10 mM MOPS, 1 M KCl, pH7.0, while the other contained an aqueous solution of 10 μMγ-cyclodextrin (γCD, Sigma) also in 10 mM MOPS, 1 M KCl, pH 7.0. The γCDbinds to WT αHL and serves as a reversible blocker, which acts as adiagnostic to show that increases in current during an applied potentialare due to pore insertion rather than current leakage through thedroplet/droplet interface.

With this material system, it was found that the time required for thelayer of lipid to form around the droplets 4 was about 30 minutes, i.e.even though lipid is expected to be adsorbed relatively quickly uponformation of the water-oil interface, it takes about 30 minutes toestablish the relatively highly dense monolayer required for bilayerformation, a density on the order of 60 Å² per molecule for thisparticular lipid. If the droplets 4 were kept separated for this timeand then brought into contact, a bilayer 14 formed but if the dropletswere left for a lesser time, the bilayer 14 was not stable and thedroplets 4 merged in less than one minute after being brought intocontact.

After formation of the bilayer 14, αHL pores inserted therein. The timerequired for bilayer formation (monitored by capacitance measurements)was between five and ten minutes. The ionic current was measured, anexample being shown in the trace of FIG. 4. The conductance of a singleαHL pore was 798±70 pS (n=6) at −50 mV, similar to a previously reportedvalue of 775 pS (5 mM HEPES, 1 M KCl, pH 7.4). The current wastransiently attenuated by γCD binding. The residence time (t_(off)) forγCD bound to the αHL pore was 495±51 ms (n=4) and was similar to theresidence time (t_(off)=421±22 ms, n=4) found in control experimentsusing a folded planar bilayer under the same solution conditions.

The form of the signal and the measurements demonstrate that thefunction of the membrane protein in the bilayer 14 is indistinguishablefrom those derived from a planar bilayer formed by the conventionalmethod. In contrast the bilayer 14 had a much greater lifetime,routinely lasting several days.

The spacing between the droplets 4 was controlled by movement of themicromanipulator 9. After formation of the bilayer 14, moving thedroplets 4 closer together causes the area of the bilayer 14 to expand,while moving the droplets 4 apart causes the area of the bilayer 14 toshrink. Although the bilayer 14 was thin enough (<5 nm) to accommodateprotein insertion, it was so stable that single channel recording waspossible even as the droplets 4 were pushed together or pulled apart,this probably being a consequence of the relatively large negative freeenergy of formation for this structure in this solvent-lipid system.

It was also demonstrated that the droplets 4 could be connected anddisconnected repeatedly, allowing examination of a protein-containingdroplet 4 many times before loss of protein activity. As an example ofthis, a droplet 4-A containing WT αHL was screened against an array ofthree analyte-containing droplets 4-B, of which: a first droplet 4contained only buffer (10 mM MOPS, 1 M KCl, pH 7.0), the second droplet4 contained 50 μM γCD in buffer, and the third droplet 4 contained 50 μMTRIMEB (a permethylated βCD) in buffer. The droplet 4-A containing WTαHL was placed on the movable cis electrode 12 supported by themicromanipulator 9 while the trans electrode 8 was common to the threeanalyte droplets 4-B, each of which was placed on one of the supports 5.

The droplet 4-A containing WT αHL was connected to the first droplet 4-Band an electrical recording was taken. Pore insertion was manifested asstepwise increases in ionic current. After recording, the droplet 4-Acontaining WT αHL was disconnected from the first droplet 4-B and movedto the second droplet 4-B, and another recording performed and so on.The transient binding of γCD (second droplet 4-B) to an αHL pore blockedaround 60% of the ionic current, while the binding of TRIMEB (thirddroplet 4-B) blocked αHL almost completely. These results show repeateduse of the droplet 4-A containing WT αHL. Furthermore after recordingfrom the third droplet 4-B, the droplet 4-A containing WT αHL wasreconnected to the first droplet 4-B and a final recording was taken.The αHL behaviour was identical to the behaviour shown in the firstscan, demonstrating that the WT αHL sample had not been contaminated byeither of the blocking analytes.

Typically, WT αHL monomer is generated by coupled in vitro transcriptionand translation (IVTT) by using an E. coli S30 extract. The protein isthen oligomerized on red blood cell membranes and purified by gelelectrophoresis. Since droplets under other circumstances can serve asnanoreactors, this idea was used to run the IVTT reaction directlyinside a droplet 4.

In particular, using the apparatus 1 of FIG. 1 two droplets 4 werebrought together. The first droplet 4 contained an IVTT mix expressingWT αHL, while the second droplet 4 contained 10 μM γCD in 2.5 mM MOPS,250 mM KCl, pH 7.0. Although the IVTT reaction is usually performed atKCl concentrations of less than 50 mM (Promega, TB129), 250 mM KCl wasadded to the IVTT mix to aid in the ionic current recordings.

In the experiments two αHL pores inserted into the bilayer. This wasobserved by the characteristic increases in the ionic current at −50 mVand by γCD binding as shown in the example trace of FIG. 5 in whichcurrent levels 0, 1 and 2 show, respectively, both pores open: one poreopen and one pore partially blocked by γCD; and both pores partiallyblocked by γCD. There is at least a two minute lag time between themixing of IVTT components and completion of the αHL polypeptide chains.Since the droplets were placed into the oil 3 within one minute ofadding the final IVTT component, the proteins must have been producedwithin the first droplet 4.

From an electrical perspective, the bilayer 14 and a pore insertedtherein are components of a circuit, in which the bilayer 14 is acapacitor, the pore is a high-resistance conductor and the ionicsolution is a wire that connects the two elements to a voltage supply.This can be used to establish the basis for more complexbio-nano-circuitry by assembling droplets 4. An example of this is nowdescribed.

Three droplets 4 were brought together in a chain, with the electrodesin the terminal droplets. The first droplet 4 contained 10 mM MOPS, 1 MKCl, pH 7.0. the second (centre) droplet 4 contained WT αHL heptamer inMOPS buffer. The third droplet 4 contained 10 μM γCD in MOPS buffer. Thefirst and third droplets 4-A were arranged on the cis and transelectrodes respectively on a support 5, while the second droplet 4-A washeld with the micromanipulator 9.

After formation of two bilayers 14 one between the first and seconddroplets 4 and the other between the second and third droplets 4, theionic current was monitored and was observed only after at least oneprotein had inserted into each bilayer 14. However, since the formationof each bilayer 14 occurred independently, it is likely that the bilayer14 which formed first began to incorporate pores before the secondbilayer 14 formed. A example current trace, which shows the binding ofγCD to the pores at the second bilayer 14, is shown in FIG. 6

The electrical properties of αHL pores and planar bilayers have beenextensively studied and are straightforward to simulate in a computermodel. In a single bilayer system, the γCD binding caused a sharpattenuation of current. However, when the γCD bound to an αHL pore atthe interface of the double bilayer system, both the binding and releasewere followed by slow current shifts to the expected levels (note thecurvature in the current trace of FIG. 6 immediately following bindingand release of γCD). A simulation of this bio-nano-circuit alsopredicted this type of binding behaviour. From the model, we attributethe slow current changes as follows. The resistance of the pore at thesecond bilayer 14 increased when the γCD bound, which lowered thepotential across the first bilayer 14. This in turn caused the firstbilayer 14 (capacitor) to release some of its stored charge. Upon γCDrelease, this process was reversed. Our results suggest that thebehaviour of chains and networks of droplets 4 is governed by basicelectrical principles and are straightforward to predict. Therefore, onemight engineer circuits to have specific functions, such as feedbackloops.

The ability to interconnect stable, compartmentalized and communicatingnanoliter volumes, or even picoliter volumes, using protein gatewaysforms the basis for the creation of a rudimentary artificial cell. Justas living cells carry out the functions of life in separatecompartments, small networks of droplets 4 might be designed to mimicthese processes. Also, just as single cells carry out functions andconjoined cells create tissue, this concept could be extended to includeepithelial and endothelial monolayers of droplet cells, where even thesize scale of 10 μm per cell is matched, allowing diffusive distances tobe equivalent. This is important because times for diffusive processesare governed by a square relationship to distance, and so droplets 4 ofdiameter 100 μm would have diffusion times that are 100 times longerthan droplets 4 of diameter 10 μm.

The αHL protein is an excellent starting point, since the properties ofthe pore, such as conductivity, ion selectivity, gating and blocking,and selective transport of small molecules can be tailored throughgenetic engineering. Further, αHL adopts a known orientation in themembrane, meaning that the direction of a chemical gradient can becontrolled through the arrangement of the droplets 4 in a network.

An ionic gradient might be combined with ion selective pores to generatea transmembrane potential and current across one droplet interface,which in turn could be used to power processes occurring at a bilayer 14farther along a chain of droplets 4.

The latter concept was demonstrated using a chain of three droplets. Thefirst droplet 4 contained N123R αHL homoheptamer (in 10 mM HEPES, 100 mMNaCl, pH 7.5) which is anion selective. The second (centre) droplet 4contained 10 mM HEPES, 1 M NaCl, pH 7.5. The third droplet 4 contained10 μM βCD and M113F/K147N αHL homoheptamer in 10 mM HEPES, 1 M NaCl, pH7.5 buffer. The first and third droplets 4 were connected electricallyto the electrodes.

After formation of two bilayers 14 one between the first and seconddroplets 4 and the other between the second and third droplets 4, theionic current was monitored. The ionic gradient across the first bilayer14 generated a potential, while the insertion of pores at both bilayers14 allowed this potential to be dissipated as ionic current. Theselectivity of the N123R pores preferentially allowed the flow of Cl−ions from the first droplet 4 to the second droplet 4, which resulted ina positive potential at first bilayer 14. It should be noted that thecircuit 15 was not used to apply a potential, rather it was only used torecord the current. As shown in the example trace of FIG. 7, the powersupplied by the battery effectively formed by the first and seconddroplets 4 enabled the observation of blocking events at the secondbilayer 14, where the M113F/K147N pores reversibly bound the molecularadapter βCD

The droplets 4 may be used to create simulated biosystems. It has beenshown that a bilayer 14 spontaneously forms and lasts several days,which might allow slow processes, such as a complete metabolic cycle, tobe studied. Proteins can be produced in situ (by IVTT) and studied bysingle-channel recording in the same droplet 4. The ability todisconnect and reconnect droplets 4 suggests that this approach might bea powerful tool in high-throughput screening and combinatorial chemistryapplications. Further, the creation of complex networks is readilyaccomplished by arranging droplets 4 in a pattern, the geometry of whichneed not be restricted to two dimensions. For example the droplets 4could be layered in say a hexagonal ABAB or ABCABC “crystal” pattern.The chain of three droplets demonstrates the feasibility of connectingnano-compartments with functional gateways and serves as a possiblestarting point for mimicking the biological hierarchy.

A network of droplets has been demonstrated as follows. Droplets 4 werecreated using a straight 20 cm section of 1.59 mm I.D. tubing that wasfilled with the oil/lipid mixture and closed at one end. With the openend up, droplets 4 were pipetted into the top of the tube just under thesurface of the oil and allowed to fall nearly to the bottom of the tube.The tube was then inverted, causing the droplet 4 to descend towards theopen end. Just before the droplet 4 reached the opening, the tube wasbrought into contact with the surface of oil 3 in a container 2 whichallowed the droplet 4 to land on the bottom of the container 2.

To form the network the base of the container had a Perspex surface witha square array of micromachined dimples (a miniature “egg-crate”), witha diameter of 1 mm and with a centre to centre spacing of 700 μm eachdimple acting as a support for a droplet 4. Electrodes were threadedthrough 200 μm diameter holes that were drilled through the bottom ofthe dimples. The underside of the cell was sealed with UV curable glueto ensure that the oil 3 did not leak around the electrodes. Allelectrodes were soldered to a common wire which was connected to theamplified (as opposed to grounded) end of a patch clamp headstage.

For visibility, each droplet 4 contained either tetramethylrhodamine(pink) or Alexa 488 linked to a dextran polymer (yellow), in 10 mM MOPS,1 M KCl, pH 7.0. When a droplet 4 was added, it formed a bilayer 4 withits neighbouring droplets 4 as the interstitial oil 3 was displaced. Theresultant network of droplets is shown in FIG. 8.

The interface between the droplets 4 was stable to mechanicalperturbation. Indeed, it was possible to puncture into a droplet 4 andthen extract it by using an agarose gel-coated Ag/AgCl electrodecontrolled by micromanipulator. Further, it was possible to replace themissing droplet 4 by stabilizing and dropping a further droplet 4 intothe empty position. This droplet spontaneously integrated into thenetwork. Thus, component droplets of the network could be extracted andexchanged without compromising the integrity of the surrounding system.

Living tissue is differentiated into regions of specific function, whichare in turn sub-differentiated into various cells. One can envisionusing the networks of droplets 4 in a similar fashion, with clusters ofdroplets 4 dedicated to certain functions. The interconnection of theseclusters might eventually lead to a rudimentary artificial tissue systemmimicing processes in living cells.

Membrane proteins may be incorporated, for example pores. The αHLprotein pore is an excellent starting point, where the pore adopts aknown orientation in the membrane, the position of protein domains(namely, the cap and stem of the pore) can be easily controlled by thearrangement of droplets 4. Further, the properties of the pore, such asunitary conductance, ion selectivity, rectification, gating,interactions with blockers and selective transport of small moleculescan be tailored through genetic engineering to provide specificfunctions in a network.

Of immediate interest are electrically propagating systems, such as theheart. Droplets containing ion gradients, gap junctions and otherproteins could be arranged in the correct order to simulate and studythe mechanism of the cardiac impulse propagation. Since the droplets 4can be disconnected and interchanged, libraries of mutant proteins couldbe screened using a functional network to study disease related proteinirregularities. For example, an ionic gradient might be combined with anion selective pore to generate a transmembrane potential across onebilayer 14, which in turn could be used to power processes occurring ata bilayer 14 farther along a chain of droplets 4 which has beendemonstrated as discussed above.

The properties of a network can be modified by changing its geometry.For example, a branched “bio-battery” was constructed as shown in FIG. 9from six 200 nl droplets 4 using the same ionic gradient as describedabove. Three droplets 4 a contained N123R αHL homoheptamer (in 10 mMHEPES, 100 Mm NaCl, pH 7.5) and were situated on the termini of a commonbranched electrode. These interfaced with three sides of an emptydroplet 4 b containing 10 mM HEPES, 1 M NaCl, pH 7.5, the remaining sideof which droplet 4 b was linked to a short chain of 10 mM HEPES, 1 MNaCl, pH 7.5 droplets 4 c containing 17 ng/ml WT αHL heptamer. Theopposing electrode was plugged into the terminal αHL droplet 4 c. Whenall droplets 4 a containing N123R αHL were connected with the terminalαHL droplet 4 c, a high current (around −390 pA) was recorded as shownin FIG. 10. As indicated by the arrow 1, one αHL droplet 4 c was thenremoved from the network, which caused the current to drop to around −61pA. As indicated by the arrow 2, removal of the second αHL droplet 4 acaused a further decreased in current to around −21 pA. As indicated bythe arrow 3, removal of the droplet 4 b stopped the current.

Nature's ability to receive and transmit information gathered fromstimuli is enabled by differentiated cells working collectively. Theretina, for example, senses light using rod and cone cells, whichinitiate a cascade of processes that transmit information down the opticnerve for interpretation by the brain. Droplet “cells” that detect lightcould be connected to droplet “cells” that conduct the current, much asin the retinal and retinal nerves. In fact other “senses” like taste andsmell that are driven by receptor binding and channel conduction evenfrom the hydrocarbon phase binding to receptors that are positioned inthe monolayers of the droplets 4 could communicate between and withinthe droplet networks. Here, receptors that are probably not natural witha hydrophobic portion bounded by hydrophilic termini, would likely notpartition or orient in a functional form in the droplet monolayer. Butone can envision a hydrophobic domain bounded by just one hydrophilicdomain that anchors the “receptor” in the monolayer facing in towardsthe water with the hydrophobic receptor in the oil phase. If such amolecule could be designed to respond to hydrophobic soluble moleculesbinding (most fragrances and active drugs are relatively hydrophobic),say by a conformational change that initiated a detectable event insidethe droplet interface, then sensing of analytes ion hydrophobic solutionmight be possible.

In a rudimentary mimic of the retina and optic nerve a light-sensingnetwork was constructed based on the light-driven proton pump,bacteriorhodopsin (BR) as shown in FIG. 11. Three droplets 4 d wereplaced on the termini of a common electrode and contained 10 mM HEPES,100 mM NaCl, pH 7.5, 0.001% dodecylmaltoside (DDM) and 18 μM BR. Acentral droplet 4 e contained 10 mM HEPES, 100 mM NaCl, pH 7.5, whilethe final outer droplet 4 f contained 10 mM HEPES, 100 mM NaCl, pH 7.5with 17 ng/ml WT αHL heptamer and had an opposing electrode plugged intoit. A 1 mW green (532 nm) pen laser was used to illuminate the network.As shown in FIG. 12, when the laser was switched on, a sharp spike incurrent was visible, which quickly decayed to around 5 pA after 5seconds. Switching the laser off caused the current to briefly dip to anegative value before returning to zero. Similar observations of BRbehaviour have been observed using analogous systems. Three cycles of 5seconds on and 5 seconds off were performed, followed by a rapidsequence of 16 laser pulses. Each BR transports one proton across themembrane per photon of light absorbed. Therefore, a 5 pA currentsuggests that tens of thousands of molecules must be functioning in themembranes 14. While such large currents might have been difficult toobtain with a single bilayer, the network of droplets 4 amplifies thelight collecting ability of the system. As a control, the droplets 4were replaced with droplets 4 containing only buffer, and the experimentwas repeated. Although the electrode surfaces were exposed to the laserduring illumination, no current from a photoelectric effect wasobserved.

In the above described experiments, the following techniques wereapplied.

Wild-type (WT), the M123R and M113F/K147N αHL heptamers were prepared byin vitro transcription and translation (IVTT), followed byoligomerization on red blood cell membranes. After purification bySDS-PAGE, the heptamer band was cut from the gel and the protein wasextracted. Typically, αHL samples were diluted between 100 to 10,000times in the buffer that was used to form the droplets 4. Afterdilution, any detergent remaining from the gel purification did notaffect the stability of the bilayer 14.

Bacteriorhodopsin (BR) from Halobacterium salinarum was purchased fromSigma. Without purification, 1 mg of BR was solubilized by sonicationfor 30 minutes in 40 μl of a one to one mixture of buffer (10 mM HEPES,100 mM NaCl, pH 7.5) and 0.01% dodecylmaltoside (DDM) in water, whichyielded a dark purple suspension. When preparing BR droplets, the stocksuspension of BR was diluted by a factor of 10 in 10 mM HEPES, 100 mMNaCl, pH 7.5.

The invention claimed is:
 1. A droplet of aqueous solution having avolume less than 1000 nL in a hydrophobic medium, the droplet comprisinga layer of amphipathic molecules around the surface of the aqueoussolution, and containing a membrane protein capable of insertion into abilayer of the amphipathic molecules.
 2. A droplet according to claim 1,wherein the membrane protein is a channel or a pore.
 3. A dropletaccording to claim 1, wherein the hydrophobic medium is an oil.
 4. Adroplet according to claim 3, wherein the oil is a hydrocarbon.
 5. Adroplet according to claim 1, wherein the amphipathic molecules arelipid molecules.
 6. A droplet according to claim 1, wherein the droplethas a volume greater than 200 nL.
 7. A droplet according to claim 1,wherein the droplet has a volume less than 800 nL.
 8. A pair ofdroplets, a first droplet of the pair of droplets being a dropletaccording to claim 1, a second droplet of the pair of droplets being adroplet of aqueous solution having a volume less than 1000 nL in thehydrophobic medium, the second droplet comprising a layer of theamphipathic molecules around the surface of the aqueous solution, andthe first droplet and the second droplet being in contact with oneanother so that a bilayer of the amphipathic molecules is formed as aninterface therebetween.
 9. A pair of droplets according to claim 8,wherein the bilayer of the amphipathic molecules has a diameter in therange from 30 μm to 1000 μm.
 10. A pair of droplets according to claim8, wherein the membrane protein is a channel or a pore.
 11. A pair ofdroplets according to claim 8, wherein the hydrophobic medium is an oil.12. A pair of droplets according to claim 11, wherein the oil is ahydrocarbon.
 13. A pair of droplets according to claim 8, wherein theamphipathic molecules are lipid molecules.
 14. A pair of dropletsaccording to claim 8, wherein the droplets each have a volume greaterthan 200 nL.
 15. A pair of droplets according to claim 8, wherein thedroplets each have a volume less than 800 nL.
 16. A pair of dropletsaccording to claim 8, wherein the second droplet contains a membraneprotein capable of insertion into a bilayer of the amphipathicmolecules.
 17. A system comprising a bilayer of amphiphilic moleculesprovided at the interface between a droplet of aqueous solution having avolume less than 1000 nL in a hydrophobic medium and a further aqueoussolution, wherein the bilayer contains a membrane protein.
 18. A systemaccording to claim 17, wherein the droplet comprises a layer ofamphipathic molecules around the surface of the aqueous solution.
 19. Adroplet according to claim 17, wherein the membrane protein is a channelor a pore.
 20. A droplet according to claim 17, wherein the hydrophobicmedium is an oil.
 21. A droplet according to claim 20, wherein the oilis a hydrocarbon.
 22. A droplet according to claim 17, wherein theamphipathic molecules are lipid molecules.
 23. A droplet according toclaim 17, wherein the droplet has a volume greater than 200 nL.
 24. Adroplet according to claim 17, wherein the droplet has a volume lessthan 800 nL.